Single-chain multivalent binding protein compositions and methods

ABSTRACT

Provided are protein, nucleic acid, and cellular libraries of single chain multivalent binding proteins (e.g., scDVD and scDVDFab molecules) and methods of using these of these libraries for the screening of single chain multivalent binding proteins using cell surface display technology (e.g., yeast display).

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/141,500, filed on Dec. 27, 2013, which claims priority from U.S.Provisional Patent Application Ser. No. 61/746,659, filed on Dec. 28,2012, which are hereby incorporated by reference in their entirety.

BACKGROUND

I. Field

The present disclosure pertains to methods and compositions forproducing single chain multivalent binding proteins that specificallybind to one or more desired target antigens. More specifically, thedisclosure relates to protein, nucleic acid, and cellular libraries ofsingle chain multivalent binding proteins (e.g., scDVD molecules) andmethods of using these libraries for the screening of single chainmultivalent binding proteins using cell surface display technology(e.g., yeast display).

II. Description of Related Art

A wide variety of multispecific antibody formats have been developed(see Kriangkum, J., et al., Biomol Eng, 2001. 18(2): p. 31-40). Amongstthem tandem single-chain Fv molecules and diabodies, and variousderivatives there of, are the most widely used formats for theconstruction of recombinant bispecific antibodies. More recentlydiabodies have been fused to Fc to generate more Ig-like molecules,named di-diabodies (see Lu, D., et al., J Biol Chem, 2004. 279(4): p.2856-65). In addition, multivalent antibody construct comprising two Fabrepeats in the heavy chain of an IgG and capable of binding four antigenmolecules has been described (see WO 0177342A1, and Miller, K., et al.,J Immunol, 2003. 170(9): p. 4854-61).

Despite the many bispecific antibody formats available to the skilledartisan, there is often a need for the skilled artisan to improve theaffinity of the bispecific antibody through affinity maturation.However, conventional affinity maturation approaches rely upon screeningfor affinity matured variants of the component binding domains of themultispecific antibody followed by their reassembly into the originalmultispecific format. Such reassembly often results in a loss of thedesired improvement in binding affinity or other desirable bindingcharacteristics. Accordingly, there is a need in the art for improvedconstructs, formats, and screening methodologies for identifyingaffinity variants of multivalent binding proteins in their desiredmultivalent format.

SUMMARY

The present disclosure provides a novel compositions and methods usefulfor the generation of improved single-chain multivalent binding proteins(e.g., scDVD) capable of binding two or more antigens simultaneouslywith high affinity.

Accordingly, in one aspect, the disclosure provides a single chainmultivalent binding protein.

In certain embodiments, the single chain multivalent binding protein hasthe general formula VH1-(X1)n-VH2-X2-VL1-(X3)n-VL2, wherein VH1 is afirst antibody heavy chain variable domain, X1 is a linker with theproviso that it is not a constant domain, VH2 is a second antibody heavychain variable domain, X2 is a linker, VL1 is a first antibody lightchain variable domain, X3 is a linker with the proviso that it is not aconstant domain, VL2 is a second antibody light chain variable domain,and n is 0 or 1, and wherein the VH1 and VL1, and the VH2 and VL2respectively combine to form two functional antigen binding sites.

In certain embodiments, the single chain binding protein has the formulaCH1-X0-VH1-(X1)n-VH2-X2-CL1-X4-VL1-(X3)n-VL2, wherein CH1 is a heavychain constant domain, X0 is a linker with the proviso that it is not aconstant domain, VH1 is a first antibody heavy chain variable domain, X1is a linker with the proviso that it is not a constant domain, VH2 is asecond antibody heavy chain variable domain, X2 is a linker, CL1 is alight chain heavy domain, X4 is a linker with the proviso that it is nota constant domain, VL1 is a first antibody light chain variable domain,X3 is a linker with the proviso that it is not a constant domain, VL2 isa second antibody light chain variable domain, and n is 0 or 1, andwherein the VH1 and VL1, and the VH2 and VL2 respectively combine toform two functional antigen binding sites. Optionally, the CL1 domaincan be a kappa (hcκ or cκ) or a lambda (hλ or cλ) constant domain. Incertain embodiments, CL1 is cκ.

In certain embodiments, X2 is a GS-rigid linker sequence. The GS rigidlinker sequence can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NOs:1-4.

In certain embodiments, the single chain multivalent binding protein hasthe general formula (VL1-(X1)n-VL2-X2-VH1-(X3)n-VH2, wherein VL1 is afirst antibody light chain variable domain, X1 is a linker with theproviso that it is not a constant domain, VL2 is a second antibody lightchain variable domain, X2 is a linker, VH1 is a first antibody heavychain variable domain, X3 is a linker with the proviso that it is not aconstant domain, VH2 is a second antibody heavy chain variable domain,and n is 0 or 1, and wherein the VH1 and VL1, and the VH2 and VL2respectively combine to form two functional antigen binding site.

In certain embodiments, the single chain binding protein has the formulaCL1-X0-VL1-(X1)n-VL2-X2-CH1-X4-VH1-(X3)n-VH2, wherein CL1 is a lightchain constant domain, X0 is a linker with the proviso that it is not aconstant domain, VL1 is a first antibody light chain variable domain, X1is a linker with the proviso that it is not a constant domain, VL2 is asecond antibody light chain variable domain, X2 is a linker, CH1 is aheavy chain constant domain, X4 is a linker with the proviso that it isnot a constant domain, VH1 is a first antibody heavy chain variabledomain, X3 is a linker with the proviso that it is not a constantdomain, VH2 is a second antibody heavy chain variable domain, and n is 0or 1, and wherein the VH1 and VL1, and the VH2 and VL2 respectivelycombine to form two functional antigen binding site. Optionally, the CL1domain can be a kappa (hcκ or cκ) or a lambda (hλ or cλ) constantdomain. In certain embodiments, CL1 is cκ.

In certain embodiments, X2 is a GS-rigid linker sequence. The GS rigidlinker sequence can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NOs:1-4.

In certain embodiments, the single chain multivalent binding protein isa single-chain dual variable domain immunoglobulin molecules (scDVD).

In certain embodiments, the single chain multivalent binding proteinfurther comprising a cell surface anchoring moiety linked to the Nand/or C terminus. In one embodiment, the anchoring moiety comprises theAga2p polypeptide.

In another aspect, the disclosure provides a polynucleotide encoding abinding protein disclosed herein.

In another aspect, the disclosure provides a host cell expressing abinding protein disclosed herein.

In another aspect, the disclosure provides a diverse library of bindingproteins.

In certain embodiments, the diverse library of binding proteinscomprises a polypeptide chain having the general formulaVH1-(X1)n-VH2-X2-VL1-(X3)n-VL2, wherein VH1 is a first heavy chainvariable domain, X1 is a linker with the proviso that it is not aconstant domain, VH2 is a second heavy chain variable domain, X2 is alinker, VL1 is a first light chain variable domain, X3 is a linker withthe proviso that it is not a constant domain, VL2 is a second lightchain variable domain, and n is 0 or 1, wherein the VH1 and VL1, and theVH2 and VL2 respectively combine to form two functional antigen bindingsites, and wherein the amino acid sequences of VH1, X1, VH2, X2, VL1,X3, and/or VL2 independently vary within the library.

In certain embodiments, the diverse library of binding proteinscomprises a polypeptide chain having the general formulaCH1-X0-VH1-(X1)n-VH2-X2-CL1-X4-VL1-(X3)n-VL2, wherein CH1 is a heavychain constant domain, X0 is a linker with the proviso that it is not aconstant domain, VH1 is a first antibody heavy chain variable domain, X1is a linker with the proviso that it is not a constant domain, VH2 is asecond antibody heavy chain variable domain, X2 is a linker, CL1 is alight chain constant domain, X4 is a linker with the proviso that it isnot a constant domain, VL1 is a first antibody light chain variabledomain, X3 is a linker with the proviso that it is not a constantdomain, VL2 is a second antibody light chain variable domain, and n is 0or 1, and wherein the VH1 and VL1, the VH2 and VL2 respectively combineto form two functional antigen binding sites, and wherein the amino acidsequences of VH1, X1, VH2, X2, VL1, X3, and/or VL2 independently varywithin the library. Optionally, the CL1 domain can be a kappa (hcκ orcκ) or a lambda (hcλ or cλ) constant domain. In certain embodiments, CL1is cκ.

In certain embodiments, X2 is a GS-rigid linker sequence. The GS rigidlinker sequence can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NOs:1-4.

In certain embodiments, the diverse library of binding proteinscomprises a polypeptide chain having the general formula(VL1-(X1)n-VL2-X2-VH1-(X3)n-VH2, wherein VL1 is a first antibody lightchain variable domain, X1 is a linker with the proviso that it is not aconstant domain, VL2 is a second antibody light chain variable domain,X2 is a linker, VH1 is a first antibody heavy chain variable domain, X3is a linker with the proviso that it is not a constant domain, VH2 is asecond antibody heavy chain variable domain, and n is 0 or 1, whereinthe VH1 and VL1, and the VH2 and VL2 respectively combine to form twofunctional antigen binding sites, and wherein the amino acid sequencesof VL1, X1, VL2, X2, VH1, X3, and/or VH2 independently vary within thelibrary.

In certain embodiments, the diverse library of binding proteinscomprises a polypeptide chain having the general formulaCL1-X0-VL1-(X1)n-VL2-X2-CH1-X4-VH1-(X3)n-VH2, wherein CL1 is a lightchain constant domain, X0 is a linker with the proviso that it is not aconstant domain, VL1 is a first antibody light chain variable domain, X1is a linker with the proviso that it is not a constant domain, VL2 is asecond antibody light chain variable domain, X2 is a linker, CH1 is aheavy chain constant domain, X4 is a linker with the proviso that it isnot a constant domain, VH1 is a first antibody heavy chain variabledomain, X3 is a linker with the proviso that it is not a constantdomain, VH2 is a second antibody heavy chain variable domain, and n is 0or 1, and wherein the VH1 and VL1, the VH2 and VL2 respectively combineto form two functional antigen binding site, and wherein the amino acidsequences of VH1, X1, VH2, X2, VL1, X3, and/or VL2 independently varywithin the library. In certain embodiments, the CL1 light chain.Optionally, the CL1 domain can be a kappa (hcκ or cκ) or a lambda (hcλor cλ) constant domain. In certain embodiments, CL1 is cκ.

In certain embodiments, X2 is a GS-rigid linker sequence. The GS rigidlinker sequence can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NOs:1-4.

In certain embodiments, each binding proteins further comprises a cellsurface anchoring moiety linked to the N or C terminus. In certainembodiments, the anchoring moiety is a cell surface protein. In oneembodiment, the anchoring moiety is Aga2p.

In certain embodiments, the polypeptide chain is a scDVD or scDVDFab.

In certain embodiments, the amino acid sequence of at least one CDR ofVH1, VH2, VL1 or VL2 independently varies within the library. In oneembodiment, the amino acid sequence of HCDR3 of VH1, VH2 independentlyvary within the library. In one embodiment, the amino acid sequence ofHCDR1 and HCDR2 of VH1 or VH2 independently vary within the library. Inone embodiment, the amino acid sequence of HCDR1, HCDR2 and HCDR3 of VH1or VH2 independently vary within the library. In one embodiment, theamino acid sequence of HCDR3 of VL1 or VL2 independently vary within thelibrary. In one embodiment, the amino acid sequence of HCDR1 and HCDR2of VL1 or VL2 independently vary within the library. In one embodiment,the amino acid sequence of HCDR1, HCDR2 and HCDR3 of VL1 or VL2independently vary within the library.

In certain embodiments, X1 independently varies within the library andwherein X1 is selected from the amino acid sequences set forth in FIG.2. In certain embodiments, X2 independently varies within the libraryand wherein X2 is (G₄S)n, where n=1-10 (SEQ ID NO: 53). In otherembodiments, X2 is selected from the amino acid sequences set forth inFIG. 11B. In specific embodiments, X2 is selected from the amino acidsequences set forth in FIG. 11B when the polypeptide chain includes CHand CL domain. In certain embodiments, X3 independently varies withinthe library and X3 is selected from the amino acid sequences set forthin FIG. 2.

In certain embodiments, the library of binding proteins share at least70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 amino acid sequence identitywith a reference binding protein. In certain embodiments, VH1 and VH2 ofthe reference binding protein specifically bind to different antigens.

In another aspect, the disclosure provides a diverse library ofpolynucleotides encoding a diverse library of binding proteins disclosedherein.

In another aspect, the disclosure provides a diverse library ofexpression vectors comprising a diverse library of polynucleotidesdisclosed herein.

In another aspect, the disclosure provides a library of transformed hostcells, expressing the diverse library of binding proteins disclosedherein.

In certain embodiments, the binding proteins are anchored on the cellsurface of a transformed host cell. In certain embodiments, the bindingproteins are anchored on the cell surface through Aga1p.

In certain embodiments, the host cells are eukaryotic. In certainembodiments, the host cells are yeast, e.g., Saccharomyces cerevisiae,Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candidatropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenulaanomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyceslactis, Kluyveromyces marxianus, Pichia pastoris, Rhodotorula rubra,Schizosaccharomyces pombe and Yarrowia lipolytica. In one embodiment,the yeast is Saccharomyces cerevisiae.

In another aspect, the disclosure provides a method of selecting abinding protein that specifically binds to a target antigen, the methodcomprising: providing a diverse library of transformed host cellsexpressing a diverse library of binding proteins disclosed herein;contacting the host cells with the target antigen; and selecting a hostcell that bind to the target antigen, thereby identifying a bindingprotein that specifically binds to a target antigen.

In another aspect, the disclosure provides a method of selecting abinding protein that specifically binds to a first and a second targetantigen simultaneously, the method comprising: providing a diverselibrary of transformed host cells expressing a diverse library ofbinding proteins disclosed herein; contacting the host cells with thefirst and second target antigen; and selecting a host cell that bind tothe first and second target antigen, thereby identifying a bindingprotein that specifically binds to a first and a second target antigensimultaneously.

In certain embodiments of the methods disclosed herein, host cells thatbind to the first and/or second antigen are selected by MagneticActivated Cell Sorting using magnetically labeled antigen. In certainembodiments of the methods disclosed herein, host cells that bind to thefirst and/or second antigen are selected by Fluorescence Activated CellSorting using fluorescently labeled antigen.

In certain embodiments, the methods disclosed herein further compriseisolating the binding protein-encoding polynucleotide sequences from theselected host cells.

In another aspect, the disclosure provides a method of producing abinding protein comprising expressing in a host cell a binding proteinthat was selected using the methods disclosed herein.

In another aspect, the disclosure provides method of producing a diverselibrary of binding proteins that specifically binds to a target antigen,the method comprising: providing a first diverse library of scDVD orscDVDFab molecules, wherein the amino acid sequence of a first region ofthe scDVD or scDVDFab molecules is varied in the library, and whereineach member of the library binds to the target antigen; providing asecond diverse library of scDVD or scDVDFab molecules, wherein the aminoacid sequence of a second region of the scDVD or scDVDFab molecules isvaried in the library, and wherein each member of the library binds tothe target antigen; recombining the first and second libraries toproduce a third diverse library of scDVD or scDVDFab molecules, whereinthe third library comprises the first regions from the first library andthe second region from the second library, thereby producing a diverselibrary of binding proteins that specifically binds to a target antigen.

In certain embodiments, the first and second libraries are recombined byyeast gap repair of polynucleotides encoding the libraries.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an exemplary single chain dual variable domain (scDVD)molecules (FIG. 1A discloses “(G₄S)_(n)” as SEQ ID NO: 54), FIG. 1Bdepicts an exemplary full-length DVD-Ig molecule, and FIG. 1C depicts anexemplary a single chain Fv molecule.

FIG. 2 is a schematic representation of an scDVD molecule and exemplaryinter-variable domain linker amino acid sequences. The linkers betweenthe VH1 and VH2 domains have amino acid sequences of SEQ ID NOs:9-30listed from top to bottom. The linkers between the VL1 and VL2 domainshave amino acid sequences of SEQ ID NOs:31-52 listed from top to bottom.FIG. 2 discloses “(G₄S)_(n)” as SEQ ID NO: 54.

FIG. 3 depicts the results of flow cytometry assays measuring the cellsurface expression of scDVD or scFv on yeast cells.

FIG. 4A depicts the results of flow cytometry assays measuring thebinding of DLL4 and/or VEGF to yeast cells expressing cell surfaceDLL4/VEGF-binding scDVD, and FIG. 4B depicts SOST and/or TNFa to yeastcells expressing cell surface SOST/TNFa-binding scDVD.

FIG. 5 depicts the results of flow cytometry assays measuring thebinding of SOST and/or TNFa to yeast cells expressing cell surfaceSOST/TNFa-binding scDVD tagged with various epitope tags.

FIG. 6A depicts the amino acid sequence of an exemplarySOST/TNFa-binding scDVD molecule (SEQ ID NO:57) (FIG. 6A discloses“(G₄S)_(n)” as SEQ ID NO: 54). FIG. 6B depicts an exemplarySOST/TNFa-binding scDVD library design, with the VH3-9, SOST VH, V1-16and MSL10VL sequences represented by SEQ ID NOs: 58-61, respectively(FIG. 6B discloses “(G₄S)_(n)” as SEQ ID NO: 54). FIG. 6C depicts theresults of flow cytometry assays measuring the binding of SOST to yeastcells expressing parental or affinity matured cell surfaceSOST/TNFa-binding scDVD. FIG. 6D depicts the results of flow cytometryassays measuring the binding of SOST to yeast cells expressing parentalor affinity matured cell surface SOST/TNFa-binding scDVD.

FIG. 7A depicts a schematic representation of an scDVD molecule andexemplary inter-VL domain linker amino acid sequences of SEQ IDNOs:62-73 listed from top to bottom (FIG. 7A discloses “(G₄S)_(n)” asSEQ ID NO: 54), and FIG. 7B depicts and results (as fold enrichment) ofyeast display screens of SOST/TNFa-binding scDVD library comprisingvarious inter-VL domain linker amino acid sequences.

FIG. 8 is a schematic representation of exemplary scDVD librariesdisclosed herein and multiplexing methods of using these libraries.

FIG. 9 is a schematic representation of exemplary scDVD librariesdisclosed herein.

FIG. 10A depicts an exemplary single chain dual variable domain Fab(scDVDFab) molecules, FIG. 10B depicts an exemplary full-length DVD-Igmolecule, and FIG. 10C depicts an exemplary a single chain DVD molecule(FIG. 10C discloses “(G₄S)_(n)” as SEQ ID NO: 54).

FIG. 11A depicts a schematic representation of an scDVDFab molecule,FIG. 11B depicts GS-rigid linker amino acid sequences (SEQ ID NOs:1-4),and FIG. 11C depicts a schematic of a scDVDFab with a GS-rigid linker(FIG. 11C discloses “G₃SG₃” as SEQ ID NO: 96 and “G₂SG₂” as SEQ ID NO:97).

FIG. 12 depicts the results of flow cytometry assays measuring theexpression of scDVDFab on the surface of yeast.

FIG. 13 depicts the results of flow cytometry assays showing that1B/IL17 scDVDFab expressed on yeast retains its ability to bind bothIL1B and/or IL17.

FIG. 14 depicts the results of flow cytometry assays showing thatscDVDFab and DVD-Fab had similar binding profiles binding to both IL1Band IL17 on the surface of yeast.

DETAILED DESCRIPTION

The present disclosure provides a novel compositions and methods usefulfor the generation of improved single-chain multivalent binding proteins(e.g., scDVD) capable of binding two or more antigens simultaneouslywith high affinity.

I. DEFINITIONS

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. Generally, nomenclature used in connectionwith, and techniques of, cell and tissue culture, molecular biology,immunology, microbiology, genetics and protein and nucleic acidchemistry and hybridization described herein are those well known andcommonly used in the art.

In order that the disclosure may be more readily understood, certainterms are first defined.

The term “multivalent binding protein” is used throughout thisspecification to denote a binding protein comprising two or more antigenbinding sites, each of which can bind independently bind to an antigen.

The terms “dual variable domain immunoglobulin” or “DVD-Ig” refer to themultivalent binding proteins disclosed in, e.g., U.S. Pat. No.8,258,268, which is herein incorporated by reference in its entirety.

The terms “single chain dual variable domain immunoglobulin” or “scDVD”refer to the antigen binding fragment of a DVD molecule that isanalogous to an antibody single chain Fv fragment. scDVD are generallyof the formula VH1-(X1)n-VH2-X2-VL1-(X3)n-VL2, where VH1 is a firstantibody heavy chain variable domain, X1 is a linker with the provisothat it is not a constant domain, VH2 is a second antibody heavy chainvariable domain, X2 is a linker, VL1 is a first antibody light chainvariable domain, X3 is a linker with the proviso that it is not aconstant domain, VL2 is a second antibody light chain variable domain,and n is 0 or 1, where the VH1 and VL1, and the VH2 and VL2 respectivelycombine to form two functional antigen binding sites. An exemplary scDVDis depicted in FIGS. 1A-1C herein.

The terms “single chain dual variable domain immunoglobulin Fab” or“scDVDFab” refer to the antigen binding fragment of a DVD molecule thatincludes the variable heavy (VH) and light (VL) chains of a DVD-Ig.scDVD are generally of the formulaCH1-X0-VH1-(X1)n-VH2-X2-CL1-X4-VL1-(X3)n-VL2, where CH1 is a heavy chainconstant domain, X0 is a linker with the proviso that it is not aconstant domain, VH1 is a first antibody heavy chain variable domain, X1is a linker with the proviso that it is not a constant domain, VH2 is asecond antibody heavy chain variable domain, X2 is a linker, CL1 is alight chain constant domain, X4 is a linker with the proviso that it isnot a constant domain, VL1 is a first antibody light chain variabledomain, X3 is a linker with the proviso that it is not a constantdomain, VL2 is a second antibody light chain variable domain, and n is 0or 1, where the VH1 and VL1, and the VH2 and VL2 respectively combine toform two functional antigen binding sites. Optionally, the CL1 domaincan be a kappa (hcκ or cκ) or a lambda (hcλ or cλ) constant domain. Incertain embodiments, CL1 is cκ. An exemplary scDVDFab is depicted inFIG. 10A, herein.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Non-limitingembodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 andIgA2) or subclass.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain and a CH3 domain, andoptionally comprises a CH4 domain. Replacements of amino acid residuesin the Fc portion to alter antibody effector function are known in theart (Winter, et al. U.S. Pat. Nos. 5,648,260; 5,624,821). The Fc portionof an antibody mediates several important effector functions e.g.cytokine induction, ADCC, phagocytosis, complement dependentcytotoxicity (CDC) and half-life/clearance rate of antibody andantigen-antibody complexes. In some cases these effector functions aredesirable for therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to Fc.gamma.Rs and complement C1q,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one amino acid residue is replaced in the constantregion of the antibody, for example the Fc region of the antibody, suchthat effector functions of the antibody are altered. The dimerization oftwo identical heavy chains of an immunoglobulin is mediated by thedimerization of CH3 domains and is stabilized by the disulfide bondswithin the hinge region (Huber et al. Nature; 264: 415-20; Thies et al1999 J Mol Biol; 293: 67-79.). Mutation of cysteine residues within thehinge regions to prevent heavy chain-heavy chain disulfide bonds willdestabilize dimeration of CH3 domains. Residues responsible for CH3dimerization have been identified (Dall'Acqua 1998 Biochemistry 37:9266-73.). Therefore, it is possible to generate a monovalent half-Ig.Interestingly, these monovalent half Ig molecules have been found innature for both IgG and IgA subclasses (Seligman 1978 Ann Immunol 129:855-70; Biewenga et al 1983 Clin Exp Immunol 51: 395-400). Thestoichiometry of FcRn: Ig Fc region has been determined to be 2:1 (Westet al 2000 Biochemistry 39: 9698-708), and half Fc is sufficient formediating FcRn binding (Kim et al 1994 Eur J Immunol; 24: 542-548.).Mutations to disrupt the dimerization of CH3 domain may not have greateradverse effect on its FcRn binding as the residues important for CH3dimerization are located on the inner interface of CH3 b sheetstructure, whereas the region responsible for FcRn binding is located onthe outside interface of CH2-CH3 domains. However the half Ig moleculemay have certain advantage in tissue penetration due to its smaller sizethan that of a regular antibody. In one embodiment at least one aminoacid residue is replaced in the constant region of the binding proteindisclosed herein, for example the Fc region, such that the dimerizationof the heavy chains is disrupted, resulting in half DVD Ig molecules.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Such antibodyembodiments may also be bispecific, dual specific, or multi-specificformats; specifically binding to two or more different antigens.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′).sub.2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Suchantibody binding portions are known in the art (Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.(ISBN 3-540-41354-5). In addition single chain antibodies also include“linear antibodies” comprising a pair of tandem Fv segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

As used herein, the terms “VH domain” and “VL domain” refer to singleantibody variable heavy and light domains, respectively, comprising FR(Framework Regions) 1, 2, 3 and 4 and CDR (Complementary DeterminantRegions) 1, 2 and 3 (see Kabat et al. (1991) Sequences of Proteins ofImmunological Interest. (NIH Publication No. 91-3242, Bethesda).

As used herein, the terms “CH1 domain” and “CL1 domain” refer to singleantibody heavy and light constant regions. A CL1 domain can be a Cκ or aCλ, domain.

As used herein, the term “CDR” or “complementarity determining region”means the noncontiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), and by Chothia et al., J. Mol. Biol.196:901-917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745(1996) where the definitions include overlapping or subsets of aminoacid residues when compared against each other. The amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth for comparison. Preferably, the term “CDR” is aCDR as defined by Kabat, based on sequence comparisons.

As used herein the term “framework (FR) amino acid residues” refers tothose amino acids in the framework region of an immunogobulin chain. Theterm “framework region” or “FR region” as used herein, includes theamino acid residues that are part of the variable region, but are notpart of the CDRs (e.g., using the Kabat definition of CDRs).

As used herein, the term “specifically binds to” refers to the abilityof a binding polypeptide to bind to an antigen with an Kd of at leastabout 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M,1×10⁻¹² M, or more, and/or bind to an antigen with an affinity that isat least two-fold greater than its affinity for a nonspecific antigen.It shall be understood, however, that the binding polypeptide arecapable of specifically binding to two or more antigens which arerelated in sequence. For example, the binding polypeptides disclosedherein can specifically bind to both human and a non-human (e.g., mouseor non-human primate) orthologs of an antigen.

The term “Polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “linker” is used to denote polypeptides comprising two or moreamino acid residues joined by peptide bonds and are used to link one ormore antigen binding portions. Such linker polypeptides are well knownin the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).Preferred linkers include, but are not limited to, the amino acidlinkers set forth in Table 7 herein.

The term “K_(on)”, as used herein, is intended to refer to the on rateconstant for association of an antibody to the antigen to form theantibody/antigen complex as is known in the art.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex as is known in the art.

The term “Kd”, as used herein, is intended to refer to the dissociationconstant of a particular antibody-antigen interaction as is known in theart.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the disclosure is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell, but, to the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.Preferably host cells include prokaryotic and eukaryotic cells selectedfrom any of the Kingdoms of life. Preferred eukaryotic cells includeprotist, fungal, plant and animal cells. Most preferably host cellsinclude but are not limited to the prokaryotic cell line E. Coli;mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; andthe fungal cell Saccharomyces cerevisiae.

II. SINGLE-CHAIN MULTIVALENT BINDING PROTEINS

In one aspect, the disclosure provides single-chain multivalent bindingproteins that can bind to two antigen simultaneously. In certainembodiments, the single-chain multivalent binding proteins generallycomprise a polypeptide of the formula VH1-(X1)n-VH2-X2-VL1-(X3)n-VL2,where VH1 is a first antibody heavy chain variable domain, X1 is alinker with the proviso that it is not a constant domain, VH2 is asecond antibody heavy chain variable domain, X2 is a linker, VL1 is afirst antibody light chain variable domain, X3 is a linker with theproviso that it is not a constant domain, VL2 is a second antibody lightchain variable domain, and n is 0 or 1, where the VH1 and VL1, and theVH2 and VL2 respectively combine to form two functional antigen bindingsites.

In certain embodiments, the single chain binding protein has the formulaCH1-X0-VH1-(X1)n-VH2-X2-CL1-X4-VL1-(X3)n-VL2, wherein CH1 is a heavychain constant domain, X0 is a linker with the proviso that it is not aconstant domain, VH1 is a first antibody heavy chain variable domain, X1is a linker with the proviso that it is not a constant domain, VH2 is asecond antibody heavy chain variable domain, X2 is a linker, CL1 is alight chain heavy domain, X4 is a linker with the proviso that it is nota constant domain, VL1 is a first antibody light chain variable domain,X3 is a linker with the proviso that it is not a constant domain, VL2 isa second antibody light chain variable domain, and n is 0 or 1, andwherein the VH1 and VL1, and the VH2 and VL2 respectively combine toform two functional antigen binding sites. Optionally, the CL1 domaincan be a kappa (hcκ or cκ) or a lambda (hλ or cλ) constant domain. Incertain embodiments, CL1 is cκ.

In certain embodiments, X2 is a GS-rigid linker sequence. The GS rigidlinker sequence can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NOs:1-4.

In certain embodiments, the single-chain multivalent binding proteinsgenerally comprise a polypeptide of the formulaVL1-(X1)n-VL2-X2-VH1-(X3)n-VH2, where VL1 is a first antibody lightchain variable domain, X1 is a linker with the proviso that it is not aconstant domain, VL2 is a second antibody light chain variable domain,X2 is a linker, VH1 is a first antibody heavy chain variable domain, X3is a linker with the proviso that it is not a constant domain, VHL2 is asecond antibody heavy chain variable domain, and n is 0 or 1, where theVH1 and VL1, and the VH2 and VL2 respectively combine to form twofunctional antigen binding sites

In certain embodiments, the single chain binding protein has the formulaCL1-X0-VL1-(X1)n-VL2-X2-CH1-X4-VH1-(X3)n-VH2, wherein CL1 is a lightchain constant domain, X0 is a linker with the proviso that it is not aconstant domain, VL1 is a first antibody light chain variable domain, X1is a linker with the proviso that it is not a constant domain, VL2 is asecond antibody light chain variable domain, X2 is a linker, CH1 is aheavy chain constant domain, X4 is a linker with the proviso that it isnot a constant domain, VH1 is a first antibody heavy chain variabledomain, X3 is a linker with the proviso that it is not a constantdomain, VH2 is a second antibody heavy chain variable domain, and n is 0or 1, and wherein the VH1 and VL1, and the VH2 and VL2 respectivelycombine to form two functional antigen binding site. Optionally, the CL1domain can be a kappa (hcκ or cκ) or a lambda (hλ or cλ) constantdomain. In certain embodiments, CL1 is cκ.

In certain embodiments, X2 is a GS-rigid linker sequence. The GS rigidlinker sequence can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NOs:1-4.

In certain embodiments, the single-chain multivalent binding proteinsare single-chain dual variable domain immunoglobulin molecules (scDVD).An exemplary scDVD is depicted in FIGS. 1A-1C herein. In otherembodiments, the single-chain multivalent binding proteins aresingle-chain dual variable domain immunoglobulin Fab molecules(scDVDFab). An exemplary scDVDFab is depicted in FIG. 10A, herein.

In certain embodiments, the multivalent binding proteins comprise a cellsurface anchoring moiety linked to the N and/or C terminus. Any moleculethat can display the binding protein on the surface of a cell can beemployed including, without limitation, cell surface protein and lipids.In certain embodiments, the anchoring moiety comprises the Aga2ppolypeptide.

The antibody variable domains for the use in the single-chainmultivalent binding proteins disclosed herein can be obtained usingrecombinant DNA techniques from a parent antibody (or DVD-Ig) generatedby any method known in the art. In a certain embodiments, the variabledomain is a murine heavy or light chain variable domain. In a certainembodiments, the variable domain is a CDR grafted or a humanizedvariable heavy or light chain domain. In a certain embodiments, thevariable domain is a human heavy or light chain variable domain.

In certain embodiments, the first and second variable domains are linkeddirectly to each other using recombinant DNA techniques. In certainembodiments, the variable domains are linked via a linker sequence.Preferably two variable domains are linked. Three or more variabledomains may also be linked directly or via a linker sequence. Thevariable domains may bind the same antigen or may bind differentantigens. Single-chain multivalent binding proteins molecules disclosedherein may include one immunoglobulin variable domain and onenon-immunoglobulin variable domain such as ligand binding domain of areceptor, active domain of an enzyme. Single-chain multivalent bindingproteins molecules may also comprise two or more non-Ig domains.

The linker sequence may be a single amino acid or a polypeptidesequence. Preferably the linker sequences are selected from the groupconsisting of consisting of the amino acid sequences set forth in FIG. 2herein.

In certain embodiments, a heavy chain or light chain constant domain islinked to the single-chain multivalent binding proteins domains usingrecombinant DNA techniques. Additionally or alternatively, in certainembodiments, the DVD heavy chain is linked to an Fc region. The Fcregion may be a native sequence Fc region, or a variant Fc region. Incertain embodiments, the Fc region is a human Fc region. In oneembodiment the Fc region includes an Fc region from IgG1, IgG2, IgG3,IgG4, IgA, IgM, IgE, or IgD.

III. LIBRARIES OF MULTIVALENT BINDING PROTEIN

In one aspect, the disclosure provides libraries of single-chainmultivalent binding proteins (e.g., scDVD molecules). Such libraries areparticularly useful for selecting multivalent binding proteins withimproved properties relative to a reference binding molecule (e.g.,improved binding kinetics or thermostability). Exemplary libraries andmethods are set forth in FIGS. 8 and 9.

In certain embodiments, the library of binding proteins comprises apolypeptide chain having the general formulaVH1-(X1)n-VH2-X2-VL1-(X3)n-VL2, wherein VH1 is a first heavy chainvariable domain, X1 is a linker with the proviso that it is not aconstant domain, VH2 is a second heavy chain variable domain, X2 is alinker, VL1 is a first light chain variable domain, X3 is a linker withthe proviso that it is not a constant domain, VL2 is a second lightchain variable domain, and n is 0 or 1, wherein the VH1 and VL1, and theVH2 and VL2 respectively combine to form two functional antigen bindingsites, and wherein the amino acid sequences of VH1, X1, VH2, X2, VL1,X3, and/or VL2 independently vary within the library. In one embodiment,the polypeptide chain is a scDVD.

In certain embodiments, the diverse library of binding proteinscomprises a polypeptide chain having the general formulaCH1-X0-VH1-(X1)n-VH2-X2-CL1-X4-VL1-(X3)n-VL2, wherein CH1 is a heavychain constant domain, X0 is a linker with the proviso that it is not aconstant domain, VH1 is a first antibody heavy chain variable domain, X1is a linker with the proviso that it is not a constant domain, VH2 is asecond antibody heavy chain variable domain, X2 is a linker, CL1 is alight chain constant domain, X4 is a linker with the proviso that it isnot a constant domain, VL1 is a first antibody light chain variabledomain, X3 is a linker with the proviso that it is not a constantdomain, VL2 is a second antibody light chain variable domain, and n is 0or 1, and wherein the VH1 and VL1, the VH2 and VL2 respectively combineto form two functional antigen binding sites, and wherein the amino acidsequences of VH1, X1, VH2, X2, VL1, X3, and/or VL2 independently varywithin the library. Optionally, the CL1 domain can be a kappa (hcκ orcκ) or a lambda (hcλ or cλ) constant domain. In certain embodiments, CL1is cκ. In one embodiment, the polypeptide chain is a scDVDFab.

In certain embodiments, X2 is a GS-rigid linker sequence. The GS rigidlinker sequence can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NOs:1-4.

In certain embodiments, the binding proteins further comprise apolypeptide chain having the general formula(VL1-(X1)n-VL2-X2-VH1-(X3)n-VH2, wherein VL1 is a first heavy chainvariable domain, X1 is a linker with the proviso that it is not aconstant domain, VL2 is a second heavy chain variable domain, X2 is alinker, VH1 is a first light chain variable domain, X3 is a linker withthe proviso that it is not a constant domain, VH2 is a second lightchain variable domain, and n is 0 or 1, wherein the VH1 and VL1, and theVH2 and VL2 respectively combine to form two functional antigen bindingsites, and wherein the amino acid sequences of VL1, X1, VL2, X2, VH1,X3, and/or VH2 independently vary within the library. In one embodiment,the polypeptide chain is a scDVD.

In certain embodiments, the diverse library of binding proteinscomprises a polypeptide chain having the general formulaCL1-X0-VL1-(X1)n-VL2-X2-CH1-X4-VH1-(X3)n-VH2, wherein CL1 is a lightchain constant domain, X0 is a linker with the proviso that it is not aconstant domain, VL1 is a first antibody light chain variable domain, X1is a linker with the proviso that it is not a constant domain, VL2 is asecond antibody light chain variable domain, X2 is a linker, CH1 is aheavy chain constant domain, X4 is a linker with the proviso that it isnot a constant domain, VH1 is a first antibody heavy chain variabledomain, X3 is a linker with the proviso that it is not a constantdomain, VH2 is a second antibody heavy chain variable domain, and n is 0or 1, and wherein the VH1 and VL1, the VH2 and VL2 respectively combineto form two functional antigen binding site, and wherein the amino acidsequences of VH1, X1, VH2, X2, VL1, X3, and/or VL2 independently varywithin the library. In certain embodiments, the CL1 light chain.Optionally, the CL1 domain can be a kappa (hcκ or cκ) or a lambda (hcλor cλ) constant domain. In certain embodiments, CL1 is cκ. In oneembodiment, the polypeptide chain is a scDVDFab.

In certain embodiments, X2 is a GS-rigid linker sequence. The GS rigidlinker sequence can comprise an amino acid sequence selected from thegroup consisting of SEQ ID NOs:1-4.

Any region of the polypeptide chains can be varied independently in thelibraries disclosed herein. In certain embodiments, the amino acidsequences of at least one CDR of VH1, VH2, VL1 or VL2 independentlyvaries within the library. In one embodiment, the amino acid sequencesof HCDR3 of VH1, VH2 independently vary within the library. In oneembodiment, the amino acid sequences of HCDR1 and HCDR2 of VH1 or VH2independently vary within the library. In one embodiment, the amino acidsequences of HCDR1, HCDR2 and HCDR3 of VH1 or VH2 independently varywithin the library. In one embodiment, the amino acid sequences of HCDR3of VL1 or VL2 independently vary within the library. In one embodiment,the amino acid sequences of HCDR1 and HCDR2 of VL1 or VL2 independentlyvary within the library. In one embodiment, the amino acid sequences ofHCDR1, HCDR2 and HCDR3 of VL1 or VL2 independently vary within thelibrary.

The linker regions X1, X2 and/or X3 can be also be varied independentlyin the libraries disclosed herein. Any length and sequence of linkerscan be employed. Suitable amino acid sequences for use in linker X1, X2and/or X3 are set forth in FIG. 2 herein. In other embodiments, X2 isselected from the amino acid sequences set forth in FIG. 11B. Inspecific embodiments, X2 is selected from the amino acid sequences setforth in FIG. 11B when the polypeptide chain includes CH and CL domain.

In certain embodiments, the libraries disclosed herein are used in cellsurface display techniques (e.g., yeast display as described in Wittrup,et al. U.S. Pat. No. 6,699,658, incorporated herein by reference).Accordingly, in certain embodiments, each binding protein in the libraryfurther comprises a cell surface anchoring moiety linked to the N and/orC terminus. Any molecule that can display the binding proteins on thesurface of a cell can be employed including, without limitation, cellsurface protein and lipids. In certain embodiments, the anchoring moietycomprise the Aga2p polypeptide.

In certain embodiments, each binding protein in the library furthercomprises an epitope tag that that can be recognized by binding protein(e.g., an antibody). Suitable tags include without limitation, includeHis, HA, c-myc, Flag, HSV, S, AcV5, E2, E, and StrepII tags.

In certain embodiments, the library of binding proteins are employed toaffinity mature a reference binding protein (e.g., scDVD or scDVDFab).Accordingly, in certain embodiments, the library of binding proteinsshare at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 amino acidsequence identity with a reference binding protein (e.g., scDVD orscDVDFab). In certain embodiments, the VH1 and VH2 of the referencebinding protein specifically bind to different antigens.

In another aspect, the disclosure provides libraries of polynucleotidesencoding the diverse library of binding proteins. The libraries can beproduced by any art recognized means. In certain embodiments, thelibraries are produced by combining portions of other libraries byoverlap PCR In certain embodiments, libraries are produced by combiningportions of other libraries by gap repair transformation in yeast cells.In certain embodiments, the nucleic acids encoding the binding proteinsare operably linked to one or more expression control elements (e.g.,promoters or enhancer elements).

In another aspect, the disclosure provides libraries of expressionvectors comprising the diverse library of polynucleotides disclosedherein. Any vectors suitable of expressing the binding proteins can beemployed.

In another aspect, the disclosure provides a library of transformed hostcells, expressing the diverse library of binding proteins disclosedherein. In certain embodiments, the individual transformed cells in thelibrary of transformed host cells express only one species from thediverse library binding proteins.

Any cells, prokaryotic or eukaryotic, are suitable for use as hostcells. In certain embodiments, the host cells are yeast including,without limitation, Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis,Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala,Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis,Kluyveromyces marxianus, Pichia pastoris, Rhodotorula rubra,Schizosaccharomyces pombe and Yarrowia lipolytica.

In certain embodiments, the expressed binding proteins are anchored onthe surface of the host cell. Any means for anchoring can be employed.In certain embodiments, the binding proteins are anchored on the cellsurface through Aga1p. This is usually achieved by the fusion of theAga2p protein the N and/or C terminus of the binding protein.

IV. SINGLE-CHAIN MULTIVALENT BINDING PROTEIN SCREENING METHODS

In another aspect, the disclosure provides a method of selecting abinding protein (e.g., scDVD or scDVDFab) that specifically binds to atarget antigen. The method generally comprises: a) providing a diverselibrary of transformed host cells expressing a diverse library ofbinding proteins disclosed herein; b) contacting the host cells with thetarget antigen; and c) selecting a host cell that bind to the targetantigen, thereby identifying a binding protein that specifically bindsto a target antigen.

In another aspect, the disclosure provides a method of selecting abinding protein that specifically binds to a first and a second targetantigen simultaneously. The method generally comprises: a) providing adiverse library of transformed host cells expressing a diverse libraryof binding proteins disclosed herein; b) contacting the host cells withthe first and second target antigen; and c) selecting a host cell thatbind to the first and second target antigen, thereby identifying abinding protein that specifically binds to a first and a second targetantigen simultaneously.

In certain embodiments of the foregoing methods, host cells that bind tothe first and/or second antigen are selected by Magnetic Activated CellSorting using magnetically labeled antigen. In certain embodiments ofthe foregoing methods, host cells that bind to the first and/or secondantigen are selected by Fluorescence Activated Cell Sorting usingfluorescently labeled antigen.

Any host cells, prokaryotic or eukaryotic, are suitable for use in theforegoing methods. In certain embodiments, the host cells are yeastincluding, without limitation, Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis,Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala,Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis,Kluyveromyces marxianus, Pichia pastoris, Rhodotorula rubra,Schizosaccharomyces pombe and Yarrowia lipolytica.

In certain embodiments, the expressed binding proteins are anchored onthe surface of the host cell. Any means for anchoring can be employed.In certain embodiments, the binding proteins are anchored on the cellsurface through Aga1p. This is usually achieved by the fusion of theAga2p protein to one or more chain of the binding protein.

After selection of antigen-binding host cells, the polynucleotidesencoding the binding proteins expressed by those cells can be isolatedusing any standard molecular biological means. These polynucleotides canbe isolated and re-expressed in another cellular or acellular system asdesired. Alternatively, these polynucleotides can be further modifiedand screened using the methods disclosed herein. In certain embodiments,the isolated polynucleotides are recombined with other polynucleotides(including libraries disclosed herein) to produce new, hybridpolynucleotides encoding novel binding proteins.

In certain embodiments, multiplex methods of screening libraries areemployed. In such methods, each individual library is barcoded by one ormore epitope tags that allows for differentiating one library or asubgroup or libraries from another library or a subgroup of libraries.Unique tag or tags are peptide sequences attached at the N-, C-, or bothtermini, or in the linker between VH and VL domains. The libraries aredifferentiated by binders (e.g., antibodies) to the epitope tags usingflow cytometry or fluorescence activated cell sorting. The method ofdifferentiation of libraries can be additive (a library having one ormore tags distinct from the others) or subtractive (a library missingone ore more tags from the others). The libraries can be kept separatelyor combined (i.e. multiplexed) for analysis or cell sorting.

In the multiplex methods, the libraries are generally introduced toorganisms that are amenable to magnetic and fluorescent activated cellsorting including, but not limited to, bacteria, yeast, and mammaliancells.

The libraries separated and distinguished by one or more tags can differaccording to one or more of the following attributes: 1) antibodygermline subgroups or sequences, light chain isotypes (kappa vs.lambda), or combinations thereof (e.g. specific VH/VL pairs); 2) naturalor synthetic (or a combination thereof) antibody or TCR sequences; 3)cell type (B, T, plasma cells, etc); 4) tissues (peripheral blood,spleen, lymph node, bone marrow, tonsil, cord blood, etc); 5) species(human, mouse, rat, llama, rabbit, chicken, hamster, shark, etc); 6)protein scaffolds (antibodies, T cell receptors, etc); ormats (antibodyand its fragments scFv, Fab, dAb, DVD-Ig, DVD-Fab, scDVD, scDVDFab,etc); 7) diversity and locations (framework vs. CDR diversity, HCDR3size and diversity, HC vs. LC diversity, DVD-Ig linkers, domainorientation, etc; and/or 8) operation logistics (operators, lablocations, cell sorters, etc)

In certain embodiments, multiple diverse libraries are created, whereeach library contains clones that vary at a different discreet region ofa reference binding protein. Each library is then screened separatelyfor binding to the desired antigen(s) and the selected clones from eachlibrary are recombined to from a new library for screening. For example,to facilitate the affinity maturation of a reference binding protein,two distinct, diverse libraries can be created: a first diverse libraryin which only the HCDR1 and HCDR2 regions of a reference antibody arevaried; and a second diverse library in which only the HCDR3 region of areference antibody are varied. The first and the second library can bescreened using the methods disclosed herein (e.g., using yeast display)to identify binding molecules with improved antigen bindingcharacteristics. The polynucleotides encoding the selected bindingproteins can then be recombined (e.g., by overlap PCR or yeast GAPrepair) to form a third library comprising the HCDR1 and HCDR2 regionsfrom the first library and the HCDR3 regions form second library. Thisthird library can then be screened using the methods disclosed herein toidentify binding proteins with further improved antigen bindingcharacteristics. Exemplary libraries and methods are set forth in FIGS.8 and 9.

Binding proteins selected using the methods disclosed herein can beisolated and re-expressed in another cellular or acellular system asdesired.

V. ENGINEERED MULTIVALENT BINDING PROTEINS

In certain preferred embodiments, the single-chain multivalent bindingproteins produced using the methods and compositions disclosed hereinexhibit improved properties (e.g., affinity or stability) with respectto a corresponding parental reference binding protein. For example, theengineered binding protein may dissociate from its target antigen with ak_(off) rate constant of about 0.1 s⁻¹ or less, as determined by surfaceplasmon resonance, or inhibit the activity of the target antigen with anIC₅₀ of about 1×10⁻⁶M or less. Alternatively, the binding protein maydissociate from the target antigen with a k_(off) rate constant of about1×10⁻²s⁻¹ or less, as determined by surface plasmon resonance, or mayinhibit activity of the target antigen with an IC₅₀ of about 1×10⁻⁷M orless. Alternatively, the binding protein may dissociate from the targetwith a k_(off) rate constant of about 1×10⁻³s⁻¹ or less, as determinedby surface plasmon resonance, or may inhibit the target with an IC₅₀ ofabout 1×10⁻⁸M or less. Alternatively, binding protein may dissociatefrom the target with a k_(off) rate constant of about 1×10⁻⁴s⁻¹ or less,as determined by surface plasmon resonance, or may inhibit its activitywith an IC₅₀ of about 1×10⁻⁹M or less. Alternatively, binding proteinmay dissociate from the target with a k_(off) rate constant of about1×10⁻⁵s⁻¹ or less, as determined by surface plasmon resonance, orinhibit its activity with an IC₅₀ of about 1×10⁻¹° M or less.Alternatively, binding protein may dissociate from the target with ak_(off) rate constant of about 1×10⁻⁵s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit its activity with an IC₅₀ ofabout 1×10⁻¹¹ M or less.

In certain embodiments, the engineered binding protein comprises a heavychain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgMor IgD constant region. Preferably, the heavy chain constant region isan IgG1 heavy chain constant region or an IgG4 heavy chain constantregion. Furthermore, the binding protein can comprise a light chainconstant region, either a kappa light chain constant region or a lambdalight chain constant region. The binding protein comprises a kappa lightchain constant region. In certain embodiments, the scDVD is reformattedinto a DVD-Ig or a DVD-Fab molecule (scDVDFab).

In certain embodiments, the engineered binding protein comprises anengineered effector function known in the art (see, e.g., Winter, et al.U.S. Pat. Nos. 5,648,260; 5,624,821). The Fc portion of a bindingprotein mediates several important effector functions e.g. cytokineinduction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC)and half-life/clearance rate of binding protein and antigen-bindingprotein complexes. In some cases these effector functions are desirablefor therapeutic binding protein but in other cases might be unnecessaryor even deleterious, depending on the therapeutic objectives. Certainhuman IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC viabinding to FcγRs and complement C1q, respectively. Neonatal Fc receptors(FcRn) are the critical components determining the circulating half-lifeof binding proteins. In still another embodiment at least one amino acidresidue is replaced in the constant region of the binding protein, forexample the Fc region of the binding protein, such that effectorfunctions of the binding protein are altered.

In certain embodiments, the engineered binding protein is derivatized orlinked to another functional molecule (e.g., another peptide orprotein). For example, a labeled binding protein disclosed herein can bederived by functionally linking a binding protein or binding proteinportion disclosed herein (by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another binding protein (e.g., a bispecific bindingprotein or a diabody), a detectable agent, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociate of the binding protein with another molecule (such as astreptavidin core region or a polyhistidine tag).

Useful detectable agents with which a binding protein or binding proteinportion disclosed herein may be derivatized include fluorescentcompounds. Exemplary fluorescent detectable agents include fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and thelike. A binding protein may also be derivatized with detectable enzymes,such as alkaline phosphatase, horseradish peroxidase, glucose oxidaseand the like. When a binding protein is derivatized with a detectableenzyme, it is detected by adding additional reagents that the enzymeuses to produce a detectable reaction product. For example, when thedetectable agent horseradish peroxidase is present, the addition ofhydrogen peroxide and diaminobenzidine leads to a colored reactionproduct, which is detectable. A binding protein may also be derivatizedwith biotin, and detected through indirect measurement of avidin orstreptavidin binding.

In other embodiment, the engineered binding protein is further modifiedto generate glycosylation site mutants in which the 0- or N-linkedglycosylation site of the binding protein has been mutated. One skilledin the art can generate such mutants using standard well-knowntechnologies. Glycosylation site mutants that retain the biologicalactivity, but have increased or decreased binding activity, are anotherobject of the present invention.

In still another embodiment, the glycosylation of the engineered bindingprotein or antigen-binding portion disclosed herein is modified. Forexample, an aglycoslated binding protein can be made (i.e., the bindingprotein lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the binding protein for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the binding protein sequence.For example, one or more amino acid substitutions can be made thatresult in elimination of one or more variable region glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the binding protein for antigen. Such anapproach is described in further detail in PCT PublicationWO2003016466A2, and U.S. Pat. Nos. 5,714,350 and 6,350,861, each ofwhich is incorporated herein by reference in its entirety.

Additionally or alternatively, an engineered binding protein disclosedherein can be further modified with an altered type of glycosylation,such as a hypofucosylated binding protein having reduced amounts offucosyl residues or a binding protein having increased bisecting GlcNAcstructures. Such altered glycosylation patterns have been demonstratedto increase the ADCC ability of binding proteins. Such carbohydratemodifications can be accomplished by, for example, expressing thebinding protein in a host cell with altered glycosylation machinery.Cells with altered glycosylation machinery have been described in theart and can be used as host cells in which to express recombinantbinding proteins disclosed herein to thereby produce a binding proteinwith altered glycosylation. See, for example, Shields, R. L. et al.(2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech.17:176-1, as well as, European Patent No: EP 1,176,195; PCT PublicationsWO 03/035835; WO 99/54342 80, each of which is incorporated herein byreference in its entirety. Using techniques known in the art apractitioner may generate binding proteins exhibiting human proteinglycosylation. For example, yeast strains have been genetically modifiedto express non-naturally occurring glycosylation enzymes such thatglycosylated proteins (glycoproteins) produced in these yeast strainsexhibit protein glycosylation identical to that of animal cells,especially human cells (U.S. patent Publication Nos. 20040018590 and20020137134 and PCT publication WO2005100584 A2).

VI. PRODUCTION OF MULTIVALENT BINDING PROTEINS

Engineered binding proteins of the present disclosure may be produced byany of a number of techniques known in the art. For example, expressionfrom host cells, wherein expression vector(s) encoding the heavy andlight chains is (are) transfected into a host cell by standardtechniques. The various forms of the term “transfection” are intended toencompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is possible toexpress the binding proteins disclosed herein in either prokaryotic oreukaryotic host cells, expression of binding proteins in eukaryoticcells is preferable, and most preferable in mammalian host cells,because such eukaryotic cells (and in particular mammalian cells) aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active binding protein.

Preferred mammalian host cells for expressing the recombinant bindingproteins disclosed herein include Chinese Hamster Ovary (CHO cells)(including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker,e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NS0 myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding binding protein genes areintroduced into mammalian host cells, the binding proteins are producedby culturing the host cells for a period of time sufficient to allow forexpression of the binding protein in the host cells or, more preferably,secretion of the binding protein into the culture medium in which thehost cells are grown. Binding proteins can be recovered from the culturemedium using standard protein purification methods.

Host cells can also be used to produce functional binding proteinfragments, such as Fab fragments or scFv molecules. It will beunderstood that variations on the above procedure are within the scopeof the present disclosure. For example, it may be desirable to transfecta host cell with DNA encoding functional fragments of either the lightchain and/or the heavy chain of a binding protein of this disclosure.Recombinant DNA technology may also be used to remove some, or all, ofthe DNA encoding either or both of the light and heavy chains that isnot necessary for binding to the antigens of interest. The moleculesexpressed from such truncated DNA molecules are also encompassed by thebinding proteins disclosed herein. In addition, bifunctional bindingproteins may be produced in which one heavy and one light chain are abinding protein disclosed herein and the other heavy and light chain arespecific for an antigen other than the antigens of interest bycrosslinking a binding protein disclosed herein to a second bindingprotein by standard chemical crosslinking methods.

In a preferred system for recombinant expression of a binding protein,or antigen-binding portion thereof, disclosed herein, a recombinantexpression vector encoding both the binding protein heavy chain and thebinding protein light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the binding protein heavy and light chain genes are eachoperatively linked to CMV enhancer/AdMLP promoter regulatory elements todrive high levels of transcription of the genes. The recombinantexpression vector also carries a DHFR gene, which allows for selectionof CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the binding protein heavyand light chains and intact binding protein is recovered from theculture medium. Standard molecular biology techniques are used toprepare the recombinant expression vector, transfect the host cells,select for transformants, culture the host cells and recover the bindingprotein from the culture medium. Still further the disclosure provides amethod of synthesizing a recombinant binding protein disclosed herein byculturing a host cell disclosed herein in a suitable culture mediumuntil a recombinant binding protein disclosed herein is synthesized. Themethod can further comprise isolating the recombinant binding proteinfrom the culture medium.

II. EXEMPLIFICATION

The present disclosure is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

Example 1. Generation of Single Chain Dual Variable Domain Molecules

The design of a scDVD molecule derived from a DVD-Ig is shownschematically in FIGS. 1A-1C. For comparison, the schematic diagrams ofa DVD-Ig (FIG. 1B) and a scFv (FIG. 1C) have also been presented. ThescDVD protein includes both the variable heavy and light chains of aDVD-Ig in their entirety with the carboxyl terminus of the VH domainstethered to the amino terminus of the VL domains through a Gly₄Serpeptide linker (SEQ ID NO: 54) of 30, 35, 40 or 45 amino acids. VH1 andVH2 are paired connected with a specific linker sequence of 6 to 14amino acids. VL1 and VL2 are paired connected with a specific linkersequence (SL) of 6 amino acids. Sequences encoding the variable regionswere PCR amplified from DVD-Ig expression vectors. Primers were designedin such a way that amplified DNAs have the necessary overlap sequence toperform additional overlapping PCRs. The final fragment contains the VHdomains, the long Gly₄Ser linker (SEQ ID NO: 54), the VL domains and apeptide tag used to monitor expression of the scDVD on the surface ofyeast. The construct is cloned by homologous recombination into a pYDyeast expression vector using DH5α chemically competent bacteria. Clonesfrom the transformation were screened by bacteria colony PCR for thepresence of the correct construct.

Several different linker sequences were evaluated for linking the VHdomains or VL domains (see FIG. 2). The SL linkers correspond to thefirst 6 to 14 amino acids amino acids of the IgG1 constant region(ASTKGPSVFPLAPS (SEQ ID NO: 55)), or corresponding to the first 6 to 14amino acids of the IgK constant region (RTVAAPSVFIFPPS (SEQ ID NO: 56)).The GS linkers correspond to 6 to 14 amino acids with repeats of Gly₄Ser(SEQ ID NO: 54). The RL linkers correspond to sequences of 6 to 14 aminoacids rich in Proline.

Example 2. scDVD Expression on the Surface of Yeast

The expression of scDVD on the surface of yeast and the suitability ofthe selected epitope tags for monitoring expression were evaluated.scDVD expression on the surface of yeast was monitored by flow cytometryanalysis using antibodies against scDVD epitope tags. The expression ofscDVD on the surface of yeast was found to be comparable to thatobserved for scFv molecules, with about 50% of the yeast cellsexpressing the scDVD construct (FIG. 3A). However, scDVD expressionshows a lower mean fluorescence intensity compared to scFv, suggesting alower number of scDVD molecules were expressed by single cell. FIG. 3A(right dot-plot) shows this difference when two different yeast cultures(one expressing scDVD and another expressing scFv) are labeled togetherin the same tube. Both constructs are expressed in about 50% of thecells (data not shown) but scFv clones have a higher mean fluorescence.

The length of the long Gly₄Ser linker (SEQ ID NO: 54) did not greatlyimpact the ability of the cells to express the scDVD. A Gly₄Ser linker(SEQ ID NO: 54) of 30 amino acids seemed to have a negative impact onthe expression while there was no difference in expression when usingGly₄Ser (SEQ ID NO: 54) of 35, 40 or 45 amino acids (FIG. 3B).

Example 3. scDVD Retains the Ability of DVD-Ig to Bind Both Targets

Two different DVD-Igs were expressed as scDVD on the surface of yeastusing pYD vectors with three different tags (AcV5, E or StrepII peptidetags). Each construct was incubated with biotinylated antigens under thesame conditions and concentrations. scDVD expression was monitored usingepitope tags specific antibodies made in mouse, goat and rabbit,respectively. Fluorochrome labeled donkey anti-mouse, goat or rabbitantibodies were used as detection reagents. Mean fluorescence is shownin each individual dot-plot. DLL4/VEGF scDVD retains its ability to bindboth DLL4 and/or VEGF (FIG. 4A). There is no difference in binding (meanfluorescence intensity) when the scDVD is incubated with DLL4, VEGF, ora mixture of the two antigens. The same findings were observed forTNF/SOST scDVD. This scDVD retains its ability to bind both TNF and/orSclerostin (FIG. 4B). There is no difference in binding (meanfluorescence intensity) when the scDVD is incubated with TNF, SOST, or amixture of the two antigens. Yeast cells express many copies of scDVD onthe cell surface, accordingly, the simultaneous binding to both antigenscould theoretically be due to some scDVD molecules on a cell binding toone antigen and other scDVD molecules on the same cell bindingindependently to the second antigen. However, the mean fluorescence donot change when the scDVD is incubated with one antigen, the otherantigen or a mix of both antigens, suggesting that the scDVD moleculesare binding both antigens simultaneously.

Example 4. scDVD Binds Both Antigens Regardless the Tag Used to Monitorits Expression on the Surface of Yeast

In yeast display, expression tags are used to monitor the antibodyexpression and to normalize the antigen-binding signal for expression,thus eliminating artifacts due to host expression bias. This allows forfine discrimination between mutants with different affinities towardstheir target. Experiments were performed to determine if any givenfunctional DVD-Ig, when expressed as a scDVD, maintains its bindingcapabilities towards its two cognate targets regardless of the tag usedto monitor its expression on the surface of yeast. Specifically,TNF/SOST DVD-Ig was expressed as scDVD on the surface of yeast usingthree different tags (AcV5, E or StrepII peptide tags). The threeconstructs were exposed to the same biotinylated antigens (TNF andSclerostin) under the same conditions and concentrations. scDVDexpression was monitored using tag-specific antibodies made in mouse(anti-AcV5; Abcam), goat (anti-E; Abcam) and rabbit (anti-StrepII;GeneScript). Fluorochrome labeled donkey anti-mouse (PerCP), goat (PE)or rabbit (DyLight488) antibodies were used as detection reagents (seeTables 1-3 herein). Antigen binding was monitored by APC conjugatedstreptavidin or Dylight633 conjugated neutravidin. All samples wereanalyzed by flow cytometry. FIG. 5 shows that it is feasible to usedifferent peptide tags to monitor scDVD expression and binding on thesurface of yeast.

Example 5. Binding Selection of a TNF/SOST scDVD Derived LibraryDemonstrate Expression and Binding Improvement Compare with the ParentalscDVD

In order to test the ability of scDVD format expressed on the surface ofyeast to enhance and affinity mature DVD-Ig, an affinity maturation of aTNF/SOST DVD-Ig was performed using different libraries. These librarieswere constructed to contain limited mutations in different CDRs of SOSTvariable domains. The TNF/SOST scDVD protein sequence is set forth inFIG. 6A. To design these libraries hypermutated CDR residues wereidentified from other human antibody sequences. The corresponding SOSTCDR residues were then subjected to limited mutagenesis by PCR withprimers having low degeneracy (79% parental nucleotide and 21% all otherthree nucleotides) at these positions to create three antibody librariesin the scDVD format suitable for yeast surface display. The firstlibrary (H1+H2) contained mutations in HCDR1 and HCDR2 of SOST VHdomain. The second library (H3) contained mutations in HCDR3 of SOST VHdomain and the third library (LC) contained mutations in all CDRs ofSOST VL domain. To further increase the identity of SOST variabledomains to the human germline framework sequence, a binary degeneracy(50% parental 50% germline) at certain positions were introduced intothe libraries and certain residues were germline (see FIG. 6B). Theintroduced changes were as follows:

H1+H2 Library:

Limited mutagenesis of residues: D30, D31, S52, H53, G54, D55, F56 andD58

Germlining 7 residues: G16R, T23A, S74A, T77S, G82bS, M87T, I89L

H3 Library:

Limited mutagenesis of residues: N95, N96, R97, G98, Y99, G100, G100a,L100b

Germlining 7 residues: G16R, T23A, S74A, T77S, G82bS, M87T, I89L

Binary degeneracy between SOST VH and germline at G94K

LC Library:

Limited mutagenesis of residues: S27, S30, T32, S40, S94

NNK randomization at residues N95a, G95b and S95c

Binary degeneracy between SOST VL and germline at G3V

These libraries (see FIG. 6B) were separately transformed and displayedon yeast cells and selected against low concentration of biotinylatedSclerostin and TNF by magnetic then fluorescence activated cell sorting.Each library was differently tagged by one of StrepII, FLAG or E peptidetags. scDVD expression and antigen binding were monitored by flowcytometry as described above using the antibodies described on Tables 2and 3 herein.

After 2 and 4 rounds of selection, the binding towards Sclerostin wasnotably improved compared to the binding of the parental molecule.Parental TNF/SOST scDVD binds to 300 nM of Sclerostin after anincubation for 1 hour at 37° C. No binding was observed when theparental molecule was incubated with 30 nM of Sclerostin. In contrast,after 2 rounds of selection the H3 library shows binding to 30 nM ofSclerostin, and after 4 round of selection the binding to 30 nM ofSclerostin is observed when the library output was incubated only for 20minutes at room temperature (see FIG. 6C). Similar improvements wereobserved for the H1+H2 and LC libraries.

Once the diversity of each library is reduced to about 10³ the plasmidDNA from each output was isolated and the libraries are recombined byPCR into a new library (rHC+LC). This library was transformed into yeastcells and displayed on cell surfaces to be selected against biotinylatedSclerostin. After selection the improvement in affinity is verynotorious. As pointed out the parental construct is able to bindSclerostin at 300 nM when incubated for 1 hour at 37° C. rHC+LC libraryoutput after 6 round of selection is able to bind 0.1 nM of Sclerostinwhen incubated only for 20 seconds at 4° C. (FIG. 6D). Although, noformal quantification of the affinity is done, an improvement of morethan 100 folds is expected based on this results. It is clear that scDVDbased libraries could be selected and enriched for better binders.

Example 6. Binding Selection of TNF/SOST scDVD Libraries ShowsEnrichment of SL Linkers Between VL Domains

As discussed above, there is a clear need for linker engineering duringthe construction and optimization of DVD-Ig antibodies. Steric hindrancedue to the proximity of the outer variable domain to the ligand bindingsite of the inner VD could, at least partially, be responsible for areduced affinity of a domain when engineered as the inner variabledomain. Accordingly, experiments were performed to determine if thescDVD approach could be used to engineer linkers to pair VHs or VLs in aDVD-Ig. To this end, a TNF/SOST scDVD library was made by introducing 12different linkers: four SL linkers corresponding to the first 6, 8, 10and 12 amino acids amino acids of the IgK constant region; four GSlinkers with repeats of Gly₄Ser (SEQ ID NO: 54) of 6, 8, 10 and 12 aminoacids; and four proline-rich RL linkers corresponding to 6, 8, 10 and 12amino acids (see FIG. 7A). Additionally, residues S94, N95a, G95b andS95c of the LCDR3 of SOST VL were mutated by NNK randomization. Afterfour rounds of selection using different concentrations of Sclerostinunder different conditions, the library output showed enrichment in RLlinkers especially of the longest size (12 and 10 amino acids; between 3to 7 folds). Also, the GS linkers were significantly reduced (between 6to 8 fold) (see FIG. 7B). This data clearly demonstrates thatscDVD-based yeast surface display allows for the optimization andengineering of linkers to pair VHs or VLs.

TABLE 1 Peptide tags used on a panel of yeast expression vectors SEQ SEQPeptide DNA ID Protein ID pYDsTEV Tag sequence NO: sequence NO: vectorsHIS* CATCATCA 74 HHHHHH 85 CCATCACC AT V5 GGTAAGCC 75 GKPIPNPL 8613767_pYDs_ TATCCCTA LGLDST TEV_total ACCCTCTC CTCGGTCT CGATTCTA CGc-MYC GAACAAAA 76 EQKLISEE 87 pYDsTEV_c-MYC ACTTATTT DL CTGAAGAA GATCTGHA TACCCATA 77 YPYDVPDY 88 pYDsTEV_HA CGATGTTC A CGGATTAC GCT HSVAGCCAGCC 78 SQPELAPE 89 pYDsTEV_HSV AGAACTCG DPED CTCCTGAA GACCCAGA GGACFLAG GACTACAA 79 DYKDDDDK 90 pYDsTEV_FLAG GGACGACG ACGACAAG StrepIITGGAGCCA 80 WSHPQFEK 91 pYDsTEV_ TCCGCAGT StrepII TTGAGAAG E2 TCCAGCAC81 SSTSSDFR 92 pYDsTEV_E2 CTCGAGTG DR ATTTTCGA GATCGC S AAGGAAAC 82KETAAAKF 93 pYDsTEV_S CGCGGCTG ERQHMDS CCAAGTTT GAACGCCA GCATATGG ATAGCE GGAGCGCC 83 GAPVPYPD 94 pYDsTEV_E TGTACCAT PLEPR ATCCGGAT CCGCTGGAACCGCGC AcV5 AGCTGGAA 84 SWKDASGW 95 pYDsTEV_AcV5 GGATGCGA S GCGGCTGGAGC *HIS tag is present in all pYDsTEV vectors downstream of all otherstags.

TABLE 2 Commercially available anti-peptide tags antibodies used tomonitor ScDVD antibody expression on yeast. Tag Ab Source Clone SourceCatalog # S Mouse SBSTAGa Abcam ab24838 S Rabbit Polyclonal ab18588 AcV5Mouse AcV5 Abcam. Rabbit S tag ab49581 antibody E2 Mouse 5E11 Abcam.AcV5 tag ab977 antibody E Rabbit Polyclonal Abcam T7 tag ® ab3397 E GoatPolyclonal Abcam ab95868 E Chicken Polyclonal ab18695 StrepII MouseStrep-tag Abcam. E tag antibody MCA2489 StrepII Rabbit Polyclonal Abcam.E tag antibody A00626 HA Mouse HA-7 Sigma H9658 HA Goat Polyclonal Abcamab9134 HA Rat (IgG1) 3F10 Roche 11-867-423 c-myc Mouse 9E10 Sigma M4439c-myc Rabbit Polyclonal Sigma C3956 Flag Mouse M2 Sigma F3165 FlagRabbit Polyclonal Sigma F7425 HSV Rabbit Polyclonal Sigma H6030

TABLE 3 Commercially available secondary reagents used to monitor scFvantibody expression and binding on the surface of yeast Secondaryreagent Fluorocrome Source Catalog # F(ab′)2 Frag. Donkey Anti-Rat IgGPerCp Jackson 712-126-150 ImmunoResearch F(ab′)2 Frag. Donkey Anti-GoatIgG R-PE Jackson ImmunoResearch F(ab′)2 Frag. Donkey Anti-Rabbit IgGDyLight-488 Jackson 705-116-147 ImmunoResearch F(ab′)2 Frag. GoatAnti-Rabbit IgG R-PE Jackson ImmunoResearch F(ab′)2 Frag. GoatAnti-Rabbit IgG Alexafluor 488 Invitrogen 711-486-152 Chicken anti mouseIgG (H + L) PerCP Jackson 111-116-144 ImmunoResearch F(ab′)2 Frag DonkeyAnti-Mouse IgG Alexafluor 633 ThermoScientific 715-126-151

Example 7. Generation of a Single Chain Dual Variable Domain Fab(scDVDFab) Including Constant Regions

Another design of a scDVDFab antibody derived from a DVD-Ig is shownschematically in FIGS. 10A-10C. For comparison, the schematic diagramsof a DVD-Ig (FIG. 10B) and a scDVD (FIG. 10C) have also been presented.In this example, the scDVDFab protein includes the variable heavy (VH)and light (VL) chains of a DVD-Ig in their entirety with the CH1 regionof the heavy chain and the kappa constant region (Cκ) of the lightchain. As shown in FIG. 10A, The VL domains fused to the Cκ are tetheredto the VH domains fused to the CH1 through a GS-rigid peptide linker of41, 49, 57 or 65 amino acids from the carboxyl terminus of the Ck regionto the amino terminus of the VH domains. These linkers are shown ingreater detail below. VL1 and VL2 are paired connected with specificlinkers already described and used in DVD-Igs and scDVD. The same is forVH1 and VH2 pair. FIG. 11A contains a schematic representation of ascDVDFab linear sequence.

Sequences encoding the variable regions were PCR amplified from theDVD-Ig expression vectors. Primers were designed in such a way thatamplified DNAs had the necessary overlap sequence to perform additionaloverlapping PCRs. The final fragment contained the linear sequencerepresented in FIG. 11A plus a peptide tag used to monitor expression ofthe scDVDFab on the surface of yeast. The construct was cloned byhomologous recombination into a pYD yeast expression vector using DH5achemically competent bacteria. Clones from the transformation werescreened by bacteria colony PCR for the presence of the right construct.

GS-Rigid Linkers

The GS-rigid linkers were made by combinations of different Gly/Sersegments and proline rich rigid segments. The sequences of the linkersare below and a GS-rigid linker scheme could be found in FIG. 11B. Morespecifically the GS-rigid linkers are composed as follows:

N-terminus-G₃SG₃-left rigid segment-G₂SG₂-right rigidsegment-G₃SG₃-C-terminus (“G₃SG₃” disclosed as SEQ ID NO: 96 and “G₂SG₂”disclosed as SEQ ID NO: 97)

where the rigid segments vary in length and amino acid composition. Thefollowing rigid segments have been tested:

Right rigid segment in the linkers: (SEQ ID NO: 98) TPAPLPAPLPT 11 AA(SEQ ID NO: 99) TPAPTPAPLPAPLPT 15 AA (SEQ ID NO: 100)TPAPLPAPTPAPLPAPLPT 19 AA (SEQ ID NO: 101) TPAPLPAPLPAPTPAPLPAPLPT 23 AALeft rigid segments in the linkers: (SEQ ID NO: 5) TPLPAPLPAPT 11 AA(SEQ ID NO: 6) TPLPTPLPAPLPAPT 15 AA (SEQ ID NO: 7)TPLPAPLPTPLPAPLPAPT 19 AA (SEQ ID NO: 8) TPLPAPLPAPLPTPLPAPLPAPT 23 AA41 aminoacids GS-rigid linker: (SEQ ID NO: 1)GGGSGGGTPLPAPLPAPTGGSGGTPAPLPAPLPTGGGSGGG 49 aminoacids GS-rigid linker:(SEQ ID NO: 2) GGGSGGGTPLPTPLPAPLPAPTGGSGGTPAPTPAPLPAPLP TGGGSGGG57 aminoacids GS-rigid linker: (SEQ ID NO: 3)GGGSGGGTPLPAPLPTPLPAPLPAPTGGSGGTPAPTPAPTP APLPAPLPTGGGSGGG65 aminoacids GS-rigid linker: (SEQ ID NO: 4)GGGSGGGTPLPAPLPAPLPTPLPAPLPAPTGGSGGTPAPTP APTPAPTPAPLPAPLPTGGGSGGG

Example 8. scDVDFab Expression on the Surface of Yeast

scDVDFab were expressed on the surface of yeast and the selected peptidetags were suitable for monitoring its expression. ScDVDFab expression onthe surface of yeast was monitored by flow cytometry analysis andantibodies were used to detect peptide tags. A DVD-Ig was expressed asscDVDFab on the surface of yeast using pYD vectors and 4 differentGS-rigid linkers. The expression of scDVDFab on the surface of yeast wascomparable to that observed for scFv molecules reaching more than 50% ofthe yeast cells expressing the construct (FIG. 12). The length of theGS-rigid linker did not impact the ability of the cells to express thescDVDFab.

Example 9. ScDVDFab Retained the Ability of DVD-Ig to Bind Both Targets

Functional DVD-Ig expressed as scDVDFab maintained its bindingcapabilities towards its two targets on the surface of yeast. A DVD-Igswas expressed as scDVDFab on the surface of yeast using pYD vectors.Aliquots of the yeast culture were incubated with biotinylated antigens.scDVDFab expression was monitored by purified tag-specific antibodies.Fluorochrome labeled secondary antibodies were used as detectionreagents. IL-1B/IL17 scDVDFab retains its ability to bind both IL1Band/or IL17 (FIG. 13).

Example 10. Binding to Both Targets is Comparable Between scDVDFab andDVD-Fab Formats Expressed on the Surface of Yeast

scDVDFab constructs bound both antigens in a similar way as the DVD-Fabbind them. A DVD-Ig was expressed as scDVDFab and DVD-Fab on the surfaceof yeast using pYD vectors. Aliquots of the yeast culture were incubatedwith biotinylated antigens. scDVDFab and DVD-Fab expression wasmonitored by purified tag-specific antibodies. Fluorochrome labeledsecondary antibodies were used as detection reagents. The scDVDFab andDVD-Fab had similar binding profiles binding to both IL1B and IL17 onthe surface of yeast. There is a small increase in the mean fluorescenceof scDVDFab compared to DVD-Fab (FIG. 14).

We claim:
 1. A single chain multivalent binding protein having thegeneral formula VH1-(X1)n-VH2-X2-VL1-(X3)n-VL2, wherein VH1 is a firstantibody heavy chain variable domain, X1 is a linker with the provisothat it is not a constant domain, VH2 is a second antibody heavy chainvariable domain, X2 is a linker, VL1 is a first antibody light chainvariable domain, X3 is a linker with the proviso that it is not aconstant domain, VL2 is a second antibody light chain variable domain,and n is 0 or 1, and wherein the VH1 and VL1, and the VH2 and VL2respectively combine to form two functional antigen binding sites.
 2. Asingle chain multivalent binding protein having the general formula(VL1-(X1)n-VL2-X2-VH1-(X3)n-VH2, wherein VL1 is a first antibody lightchain variable domain, X1 is a linker with the proviso that it is not aconstant domain, VL2 is a second antibody light chain variable domain,X2 is a linker, VH1 is a first antibody heavy chain variable domain, X3is a linker with the proviso that it is not a constant domain, VH2 is asecond antibody heavy chain variable domain, and n is 0 or 1, andwherein the VH1 and VL1, and the VH2 and VL2 respectively combine toform two functional antigen binding site.
 3. The binding protein ofclaim 1 or 2 which is a single-chain dual variable domain immunoglobulinmolecules (scDVD).
 4. The binding protein of any one of the precedingclaims, further comprising a cell surface anchoring moiety linked to theN and/or C terminus.
 5. The binding protein of claim 4, wherein theanchoring moiety comprises the Aga2p polypeptide.
 6. A polynucleotideencoding a binding protein of any one of the preceding claims.
 7. A hostcell expressing a binding protein of any one of the preceding claims. 8.A diverse library of binding proteins comprising a polypeptide chainhaving the general formula VH1-(X1)n-VH2-X2-VL1-(X3)n-VL2, wherein VH1is a first heavy chain variable domain, X1 is a linker with the provisothat it is not a constant domain, VH2 is a second heavy chain variabledomain, X2 is a linker, VL1 is a first light chain variable domain, X3is a linker with the proviso that it is not a constant domain, VL2 is asecond light chain variable domain, and n is 0 or 1, wherein the VH1 andVL1, and the VH2 and VL2 respectively combine to form two functionalantigen binding sites, and wherein the amino acid sequences of VH1, X1,VH2, X2, VL1, X3, and/or VL2 independently vary within the library.
 9. Adiverse library of binding proteins comprising a polypeptide chainhaving the general formula (VL1-(X1)n-VL2-X2-VH1-(X3)n-VH2, wherein VL1is a first antibody light chain variable domain, X1 is a linker with theproviso that it is not a constant domain, VL2 is a second antibody lightchain variable domain, X2 is a linker, VH1 is a first antibody heavychain variable domain, X3 is a linker with the proviso that it is not aconstant domain, VH2 is a second antibody heavy chain variable domain,and n is 0 or 1, wherein the VH1 and VL1, and the VH2 and VL2respectively combine to form two functional antigen binding sites, andwherein the amino acid sequences of VL1, X1, VL2, X2, VH1, X3, and/orVH2 independently vary within the library.
 10. The diverse library ofclaim 8 or 9, wherein each binding proteins further comprises a cellsurface anchoring moiety linked to the N or C terminus.
 11. The diverselibrary of claim 10, wherein the anchoring moiety is a cell surfaceprotein.
 12. The diverse library of claim 10, wherein the anchoringmoiety is Aga2p.
 13. The diverse library of any one of the precedingclaims, wherein the polypeptide chain is a scDVD.
 14. The library of anyone of the preceding claims, wherein the amino acid sequence of at leastone CDR of VH1, VH2, VL1 or VL2 independently varies within the library.15. The library of any one of the preceding claims, wherein the aminoacid sequence of HCDR3 of VH1, VH2 independently vary within thelibrary.
 16. The library of any one of the preceding claims, wherein theamino acid sequence of HCDR1 and HCDR2 of VH1 or VH2 independently varywithin the library.
 17. The library of any one of the preceding claims,wherein the amino acid sequence of HCDR1, HCDR2 and HCDR3 of VH1 or VH2independently vary within the library.
 18. The library of any one of thepreceding claims, wherein the amino acid sequence of HCDR3 of VL1 or VL2independently vary within the library.
 19. The library of any one of thepreceding claims, wherein the amino acid sequence of HCDR1 and HCDR2 ofVL1 or VL2 independently vary within the library.
 20. The library of anyone of the preceding claims, wherein the amino acid sequence of HCDR1,HCDR2 and HCDR3 of VL1 or VL2 independently vary within the library. 21.The library of any one of the preceding claims, wherein X1 independentlyvaries within the library and wherein X1 is selected from the amino acidsequences set forth in FIG.
 2. 22. The library of any one of thepreceding claims, wherein X2 independently varies within the library andwherein X2 is (G₄S)n, where n=1-10.
 23. The library of any one of thepreceding claims, wherein X3 independently varies within the library andwherein X3 is selected from the amino acid sequences set forth in FIG.2.
 24. The library of any one of the preceding claims, wherein thelibrary of binding proteins share at least 70, 75, 80, 85, 90, 95, 96,97, 98, or 99 amino acid sequence identity with a reference bindingprotein.
 25. The library of any one of the preceding claims, wherein VH1and VH2 of the reference binding protein specifically bind to differentantigens.
 26. A diverse library of polynucleotides encoding the diverselibrary of binding proteins of any one of the preceding claims.
 27. Adiverse library of expression vectors comprising the diverse library ofpolynucleotides of claim
 26. 28. A library of transformed host cells,expressing the diverse library of binding proteins of any one of thepreceding claims.
 29. The library of transformed host cells of claim 28,wherein the binding proteins are anchored on the cell surface.
 30. Thelibrary of transformed host cells of claim 28, wherein the bindingproteins are anchored on the cell surface through Aga1p.
 31. The libraryof transformed host cells of claim 28, wherein the host cells areeukaryotic.
 32. The library of transformed host cells of claim 31,wherein the host cells are yeast.
 33. The library of transformed hostcells of claim 31, wherein the yeast is selected from the groupconsisting of Saccharomyces cerevisiae, Saccharomyces carlsbergensis,Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcuslaurentii, Cryptococcus neoformans, Hansenula anomala, Hansenulapolymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromycesmarxianus, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombeand Yarrowia lipolytica.
 34. The library of transformed host cells ofclaim 31, wherein the yeast is Saccharomyces cerevisiae.
 35. A method ofselecting a binding protein that specifically binds to a target antigen,the method comprising: a) providing a diverse library of transformedhost cells expressing the diverse library of binding proteins of any oneof claims 8-25; b) contacting the host cells with the target antigen;and c) selecting a host cell that bind to the target antigen, therebyidentifying a binding protein that specifically binds to a targetantigen.
 36. A method of selecting a binding protein that specificallybinds to a first and a second target antigen simultaneously, the methodcomprising: a) providing a diverse library of transformed host cellsexpressing the diverse library of binding proteins of any one of claims8-25; b) contacting the host cells with the first and second targetantigen; and c) selecting a host cell that bind to the first and secondtarget antigen, thereby identifying a binding protein that specificallybinds to a first and a second target antigen simultaneously.
 37. Themethod of claim 35 or 36, wherein host cells that bind to the firstand/or second antigen are selected by Magnetic Activated Cell Sortingusing magnetically labeled antigen.
 38. The method of claim 35, 36, or37, wherein host cells that bind to the first and/or second antigen areselected by Fluorescence Activated Cell Sorting using fluorescentlylabeled antigen.
 39. The method of any one of claims 35-38, furthercomprising isolating the binding protein-encoding polynucleotidesequences from the host cells selected in step (c).
 40. A method ofproducing a binding protein, comprising expressing in a host cell abinding protein that was selected using the methods of any of claims8-25.
 41. A method of producing a diverse library of binding proteinsthat specifically binds to a target antigen, the method comprising: a)providing a first diverse library of scDVD molecules, wherein the aminoacid sequence of a first region of the scDVD molecules is varied in thelibrary, and wherein each member of the library binds to the targetantigen; b) providing a second diverse library of scDVD molecules,wherein the amino acid sequence of a second region of the scDVDmolecules is varied in the library, and wherein each member of thelibrary binds to the target antigen; c) recombining the first and secondlibraries to produce a third diverse library of scDVD molecules, whereinthe third library comprises the first regions from the first library andthe second region from the second library, thereby producing a diverselibrary of binding proteins that specifically binds to a target antigen.42. The method of claim 41, wherein the first and second libraries arerecombined by yeast gap repair of polynucleotides encoding thelibraries.